Integrin mobilizes intracellular Ca(2+) in renal vascular smooth muscle cells

Peptides with the Arg-Gly-Asp (RGD) motif induce vasoconstriction in rat afferent arterioles by increasing the intracellular Ca(2+) concentration ([Ca(2+)](i)) in vascular smooth muscle cells (VSMC). This finding suggests that occupancy of integrins on the plasma membrane of VSMC might affect vascular tone. The purpose of this study was to determine whether occupancy of integrins by exogenous RGD peptides initiates intracellular Ca(2+) signaling in cultured renal VSMC. When smooth muscle cells were exposed to 0.1 mM hexapeptide GRGDSP, [Ca(2+)](i) rapidly increased from 91 +/- 4 to 287 +/- 37 nM and then returned to the baseline within 20 s (P < 0.05, 34 cells/5 coverslips). In controls, the hexapeptide GRGESP did not trigger Ca(2+) mobilization. Local application of the GRGDSP induced a regional increase of cytoplasmic [Ca(2+)](i), which propagated as Ca(2+) waves traveling across the cell and induced a rapid elevation of nuclear [Ca(2+)](i). Spontaneous recurrence of smaller-amplitude Ca(2+) waves were found in 20% of cells examined after the initial response to RGD-containing peptides. Blocking dihydropyridine-sensitive Ca(2+) channels with nifedipine or removal of extracellular Ca(2+) did not inhibit the RGD-induced Ca(2+) mobilization. However, pretreatment of 20 microM ryanodine completely eliminated the RGD-induced Ca(2+) mobilization. Anti-beta(1) and anti-beta(3)-integrin antibodies with functional blocking capability simulate the effects of GRGDSP in [Ca(2+)](i). Incubation with anti-beta(1)- or beta(3)-integrin antibodies inhibited the increase in [Ca(2+)](i) induced by GRGDSP. We conclude that exogenous RGD-containing peptides induce release of Ca(2+) from ryanodine-sensitive Ca(2+) stores in renal VSMC via integrins, which can trigger cytoplasmic Ca(2+) waves propagating throughout the cell.     

Light sensitivity of the ciliate Tetrahymena vorax induced by the fluorescent dye acridine orange.

The ciliate Tetrahymena vorax is normally insensitive to light. However, after uptake of acridine orange, blue light evokes instant backward swimming. The dye accumulates mainly in posterior vacuoles, with half-maximal uptake after 1 min. Illumination for 10 s induced a depolarisation of approximately 15 mV lasting less than 2 s, followed by a sustained hyperpolarisation of approximately 20 mV. Deciliated cells displayed a similar response. The hyperpolarisation was linked to reduced membrane resistance, showed a reversal potential of approximately -55 mV and was blocked by 1 mmol l(-1) TEA. The rate of rise of electrically evoked Ca(2+)-spikes was reduced during the hyperpolarisation, which is compatible with elevated cytosolic Ca(2+) concentration. This suggests that the hyperpolarisation may be caused by activation of Ca(2+)-sensitive K(+) channels. The depolarisation was abolished in Ca(2+)-free medium, whereas the hyperpolarisation was unaffected. Illumination for 2 s, or prolonged stimulation restricted to the anterior part of the cell, induced depolarisation only. Illumination of the posterior part caused delayed hyperpolarisation with no preceding depolarisation. We conclude that the induced backward swimming is associated with Ca(2+) influx through anterior channels, while Ca(2+) released from intracellular stores activates K(+) channels responsible for the delayed hyperpolarisation.     

Nongenomic mechanism of glucocorticoid inhibition of bradykinin-induced calcium influx in PC12 cells: possible involvement of protein kinase C

Many stimulants, including bradykinin (BK), can induce increase in [Ca(2+)](i) in PC12 cells. Bradykinin induces an increase in [Ca(2+)](i) via intracellular Ca(2+) release and extracellular Ca(2+) influx through the transduction of G protein, but not through voltage-sensitive calcium channels. In this experiment, We analyzed how corticosterone (Cort) influences BK-induced intracellular Ca(2+) release and extracellular Ca(2+) influx, and further studied the mechanism of glucocorticoid's action. To dissociate the intracellular Ca(2+) release and extracellular Ca(2+) influx induced by BK, the Ca(2+)-free/Ca(2+)- reintroduction protocol was used. The results were as follows: (1) The Ca(2+) influx induced by BK could be rapidly inhibited by Cort, but intracellular Ca(2+) release could not be affected significantly. (2) The inhibitory effect of Cort-BSA (BSA -conjugated Cort) on Ca(2+) influx induced by BK was the same as the effect of free Cort. (3) Protein kinase C (PKC) activator (phorbol 12-myristate 13-acetate) could mimic and PKC inhibitor G?6976 could reverse the inhibitory effect of Cort. (4) There was no inhibitory effect of Cort on Ca(2+) influx induced by BK when pretreated with pertussis toxin. The results suggested, for the first time, that Cort might act via a putative membrane receptor and inhibit the Ca(2+) influx induced by BK through the pertussis toxin -sensitive G protein-PKC pathway.     

Intercellular calcium waves in HeLa cells expressing GFP-labeled connexin 43, 32, or 26

This study was undertaken to obtain direct evidence for the involvement of gap junctions in the propagation of intercellular Ca(2+) waves. Gap junction-deficient HeLa cells were transfected with plasmids encoding for green fluorescent protein (GFP) fused to the cytoplasmic carboxyl termini of connexin 43 (Cx43), 32 (Cx32), or 26 (Cx26). The subsequently expressed GFP-labeled gap junctions rendered the cells dye- and electrically coupled and were detected at the plasma membranes at points of contact between adjacent cells. To correlate the distribution of gap junctions with the changes in [Ca(2+)](i) associated with Ca(2+) waves and the distribution of the endoplasmic reticulum (ER), cells were loaded with fluorescent Ca(2+)-sensitive (fluo-3 and fura-2) and ER membrane (ER-Tracker) dyes. Digital high-speed microscopy was used to collect a series of image slices from which the three-dimensional distribution of the gap junctions and ER were reconstructed. Subsequently, intercellular Ca(2+) waves were induced in these cells by mechanical stimulation with or without extracellular apyrase, an ATP-degrading enzyme. In untransfected HeLa cells and in the absence of apyrase, cell-to-cell propagating [Ca(2+)](i) changes were characterized by initiating Ca(2+) puffs associated with the perinuclear ER. By contrast, in Cx-GFP-transfected cells and in the presence of apyrase, [Ca(2+)](i) changes were propagated without initiating perinuclear Ca(2+) puffs and were communicated between cells at the sites of the Cx-GFP gap junctions. The efficiency of Cx expression determined the extent of Ca(2+) wave propagation. These results demonstrate that intercellular Ca(2+) waves may be propagated simultaneously via an extracellular pathway and an intracellular pathway through gap junctions and that one form of communication may mask the other.     

Cadherins mediate intercellular mechanical signaling in fibroblasts by activation of stretch-sensitive calcium-permeable channels

Cells in mechanically active environments form extensive, cadherin-mediated intercellular junctions that are important in tissue remodeling and differentiation. Currently, it is unknown whether adherens junctions in connective tissue fibroblasts transmit mechanical signals and coordinate multicellular adaptations to physical forces. We hypothesized that cadherins mediate intercellular mechanotransduction by activating calcium-permeable, stretch-sensitive channels. Human gingival fibroblasts in suspension were plated on established homotypic monolayer cultures. The cells formed intercellular adherens junctions. Controlled mechanical forces were applied to intercellular junctions by electromagnets acting on cells containing internalized magnetite beads. At early but not later stages of intercellular attachment, force application visibly displaced magnetite bead-loaded cells and induced robust Ca(2+) transients (65 +/- 9.4 nm above base line). Similar Ca(2+) transients were induced by force application to anti-N-cadherin antibody-coated magnetite beads. Ca(2+) responses depended on influx of extracellular Ca(2+) through mechanosensitive channels because both Ca(2+) chelation and gadolinium chloride abolished the response and MnCl(2) quenched fura-2 fluorescence after force application. Force application induced accumulation of microinjected rhodamine-actin at intercellular contacts; actin assembly was inhibited by buffering intracellular calcium fluxes. Our results indicate that mechanical forces applied to adherens junctions activate stretch-sensitive calcium-permeable channels and increase actin polymerization. We suggest that N-cadherins in fibroblasts are intercellular mechanotransducers.     

Subcellular organization of agonist-evoked Ca(2+) waves in the blowfly salivary gland

We have studied the subcellular organization of intra- and intercellular Ca(2+)waves elicited by the neurohormone 5-hydroxytryptamine (5-HT) in intact blowfly salivary glands by using Ca(2+)-sensitive fluorescent probes and confocal microscopy. 5-HT (3 nM) elicited repetitive Ca(2+)waves (1) that were initiated at Ca(2+)-release sites close to the basal plasma membrane, (2) that sequentially spread to the cell apex and (3) that, after a delay of 0.7 +/- 0.20 s at the cell boundaries, spread into adjacent cells. [Ca(2+)](i)increases in the adjacent cells were first detectable at those portions of the lateral plasma membrane that faced a previously activated cell. Electron microscopy revealed that the sites of Ca(2+)wave transmission between the cells are correlated with the distribution of gap junctions that cluster in the basal cell portions. The ensuing intracellular Ca(2+)wave propagated at constant velocity (27 +/- 7.3 microm/s) in the lateral cell plane. Moreover, a basally to apically propagating wavefront was detectable at the cell membrane that bordered on the neighbor that provided the excitatory signal, whereas [Ca(2+)](i)increased simultaneously both apically and basally at the opposite lateral cell border. Overall, the subcellular patterns of Ca(2+)wave propagation differed from the patterns observed in mammalian secretory epithelial cells. The findings impose some constraints on the functional significance of intra- and intercellular Ca(2+)waves and potential mechanisms underlying 5-HT-evoked fluid secretion.     

Intercellular calcium signaling in astrocytes via ATP release through connexin hemichannels.

Astrocytes are capable of widespread intercellular communication via propagated increases in intracellular Ca(2+) concentration. We have used patch clamp, dye flux, ATP assay, and Ca(2+) imaging techniques to show that one mechanism for this intercellular Ca(2+) signaling in astrocytes is the release of ATP through connexin channels ("hemichannels") in individual cells. Astrocytes showed low Ca(2+)-activated whole-cell currents consistent with connexin hemichannel currents that were inhibited by the connexin channel inhibitor flufenamic acid (FFA). Astrocytes also showed molecular weight-specific influx and release of dyes, consistent with flux through connexin hemichannels. Transmembrane dye flux evoked by mechanical stimulation was potentiated by low Ca(2+) and was inhibited by FFA and Gd(3+). Mechanical stimulation also evoked release of ATP that was potentiated by low Ca(2+) and inhibited by FFA and Gd(3+). Similar whole-cell currents, transmembrane dye flux, and ATP release were observed in C6 glioma cells expressing connexin43 but were not observed in parent C6 cells. The connexin hemichannel activator quinine evoked ATP release and Ca(2+) signaling in astrocytes and in C6 cells expressing connexin43. The propagation of intercellular Ca(2+) waves in astrocytes was also potentiated by quinine and inhibited by FFA and Gd(3+). Release of ATP through connexin hemichannels represents a novel signaling pathway for intercellular communication in astrocytes and other non-excitable cells.     

Depolarisation induces rapid and transient formation of intracellular sphingosine-1-phosphate.

Formation of sphingosine-1-phosphate (SPP) by sphingosine kinase serves as a signalling pathway for various membrane receptors. Here, we show that membrane depolarisation is another mechanism by which this pathway can be activated. Formation of [(3)H]SPP as well as levels of endogenous SPP were rapidly and transiently increased in PC12 pheochromocytoma cells depolarised with high KCl. Time course and maximum were similar to those induced by bradykinin. Depolarisation-induced SPP production was also observed in RINm5F insulinoma cells, dependent on extracellular Ca(2+) and fully suppressed by verapamil, thus apparently caused by Ca(2+) influx via voltage-gated Ca(2+) channels. Studies with sphingosine kinase inhibitors and overexpression of sphingosine kinase revealed a partial contribution of this pathway to depolarisation-induced noradrenaline release and Ca(2+) increase.     

Slow intercellular Ca(2+) signaling in wild-type and Cx43-null neonatal mouse cardiac myocytes

Focal mechanical stimulation of single neonatal mouse cardiac myocytes in culture induced intercellular Ca(2+) waves that propagated with mean velocities of approximately 14 micrometer/s, reaching approximately 80% of the cells in the field. Deletion of connexin43 (Cx43), the main cardiac gap junction channel protein, did not prevent communication of mechanically induced Ca(2+) waves, although the velocity and number of cells communicated by the Ca(2+) signal were significantly reduced. Similar effects were observed in wild-type cardiac myocytes treated with heptanol, a gap junction channel blocker. Fewer cells were involved in intercellular Ca(2+) signaling in both wild-type and Cx43-null cultures in the presence of suramin, a P(2)-receptor blocker; blockage was more effective in Cx43-null than in wild-type cells. Thus gap junction channels provide the main pathway for communication of slow intercellular Ca(2+) signals in wild-type neonatal mouse cardiac myocytes. Activation of P(2)-receptors induced by ATP release contributes a secondary, extracellular pathway for transmission of Ca(2+) signals. The importance of such ATP-mediated Ca(2+) signaling would be expected to be enhanced under ischemic conditions, when release of ATP is increased and gap junction channels conductance is significantly reduced.     

Crosstalk between cytosolic pH and intracellular calcium in human lymphocytes: effect of 4-aminopyridin, ammoniun chloride and ionomycin

Stimulation of lymphocytes by specific antigens is followed by the activation of different signal transduction mechanisms, such as alterations in the cytoplasmic levels of Ca(2+), H(+) and variations in membrane potential. To study interrelationships among these parameters, changes in pHi and Ca(2+) were measured with the fluorescent probes BCECF and Fura-2 in freshly isolated blood human lymphocytes. Moreover, membrane potential qualitative alterations were recorded with the fluorescent dye bis-oxonol. In a bicarbonate-free medium, cell alkalinization with NH(4)Cl slightly decreased intracellular Ca(2+) concentration ([Ca(2+)](i)) due to efflux of Ca(2+) from the cell. In contrast, an elevation of pHi induced with 4-AP increased [Ca(2+)](i), either in the presence or absence of external Ca(2+). The increase in Ca(2+)-free medium is likely to be due to Ca(2+) release from thapsigargin and caffeine-independent intracellular stores. Both 4-AP or NH(4)Cl induced a plasma membrane depolarisation, although with different kinetics. The ionosphere ionomycin increased pHi, Ca(2+) levels and also induced membrane depolarisation. Together, these observations demonstrate a lack of correlation between the magnitude of changes in pHi and Ca(2+).     

Cholesterol depletion inhibits store-operated calcium currents and exocytotic membrane fusion in RBL-2H3 cells

The effects of cholesterol depletion from the plasma membrane with methyl-beta-cyclodextrin (MbetaCD) on exocytotic processes were investigated in rat basophil leukemia cells (RBL-2H3 cells). Pretreatment of the cells with MbetaCD inhibited antigen-evoked exocytotic release dose-dependently. To elucidate the mechanism of this inhibition, we performed experiments on the effects of MbetaCD on exocytotic membrane fusion and mobilization of Ca(2+) and on the localization of the tyrosine kinase Lyn. Inhibition of degranulation by MbetaCD was observed even under stimulation with the phorbol ester and calcium ionophore. Therefore, MbetaCD affected a process downstream of Ca(2+) influx, or membrane fusion between the granule and the plasma membrane. Intracellular calcium measurements revealed that MbetaCD inhibited the Ca(2+) increase induced by antigen. Furthermore, we found that MbetaCD significantly inhibited Ca(2+) influx from the extracellular medium through the store-operated calcium channel (SOC) but did not affect Ca(2+) release from the intracellular Ca(2+) store. Fluorescent image analysis of cells expressing Lyn-YFP showed that treatment with MbetaCD scarcely affected the localization and lateral mobility of Lyn in the plasma membrane. These results suggest that cholesterol depletion by MbetaCD decreases degranulation mainly by inhibiting the SOC and membrane fusion between the secretory granules and the plasma membrane in mast cells.     

Lipid rafts function in Ca2+ signaling responsible for activation of sperm motility and chemotaxis in the ascidian Ciona intestinalis

Lipid rafts are specialized membrane microdomains that function as signaling platforms across plasma membranes of many animal and plant cells. Although there are several studies implicating the role of lipid rafts in capacitation of mammalian sperm, the function of these structures in sperm motility activation and chemotaxis remains unknown. In the ascidian Ciona intestinalis, egg-derived sperm activating- and attracting-factor (SAAF) induces both activation of sperm motility and sperm chemotaxis to the egg. Here we found that a lipid raft disrupter, methyl-β-cyclodextrin (MCD), inhibited both SAAF-induced sperm motility activation and chemotaxis. MCD inhibited both SAAF-promoted synthesis of intracellular cyclic AMP and sperm motility induced by ionophore-mediated Ca(2+) entry, but not that induced by valinomycin-mediated hyperpolarization. Ca(2+)-imaging revealed that lipid raft disruption inhibited Ca(2+) influx upon activation of sperm motility. The Ca(2+)-activated adenylyl cyclase was clearly inhibited by MCD in isolated lipid rafts. The results suggest that sperm lipid rafts function in signaling upstream of cAMP synthesis, most likely in SAAF-induced Ca(2+) influx, and are required for Ca(2+)-dependent pathways underlying activation and chemotaxis in Ciona sperm.     

肾上腺髓质素降低培养海马神经元胞内游离钙离子浓度

经荧光探针Fluo 3-AM标记细胞内游离钙后,用激光共聚焦显微镜检测肾上腺髓质素(adrenomedullin,ADM)对原代培养大鼠海马神经元内游离钙浓度([Ca^2 ]1)的影响。实验结果如下：(1)ADM(0．01-1．0μmol／L)浓度依赖性地降低细胞内钙浓度。(2)降钙素基因相关肽受体阻断剂(calcitonin gene-related peptide,CGRP8-37)预处理可部分抑制ADM的效应。(3)ADM可显著抑制高钾引起的[Ca^2 ]1增加。(4)ADM可显著抑制三磷酸肌醇(inositol 1,4,5-trisphosphate,IP3)引起的内钙释放,而对兰尼定(ryanodine)引起的内钙释放无显著影响。以上结果提示,ADM降低培养海马神经元内游离钙浓度,此作用与其抑制IP,引起的内钙释放有关,ADM对静息状态下的Ca^2 内流无影响,但可显著抑制高钾引起的Ca^2 内流,CGRP受体介导了ADM的上述效应。     

GluR2 knockdown reveals a dissociation between [Ca2+]i surge and neurotoxicity

Reduction in GluR2 subunit expression and subsequent increases in AMPA receptor mediated Ca(2+) currents were postulated to exacerbate glutamate neurotoxicity following seizures or global ischemia. To directly test the effects of shifting the GluR1/GluR2 subunit ratio on excitotoxicity, GluR2 antisense deoxyoligonucleotides (AS-ODNs) were applied to dissociated hippocampal cultures for 1-8 days. The GluR1/GluR2 protein ratio was examined immunohistochemically and by Western blotting. [Ca(2+)](i) concentrations were determined by ratiometric imaging of Fura 2-loaded cells. The cultures were exposed to glutamate, AMPA, NMDA or kainic acid (KA) 3 days after GluR2 knockdown and cell viability was determined 1 day later by MTT reduction assay or Trypan blue exclusion. Although GluR2 AS-ODNs increased the GluR1/GluR2 protein ratio in a time dependent manner, neurons and glia appeared healthy and MTT reduction values were similar to untreated and sense controls. Basal [Ca(2+)](i) levels were unchanged but [Ca(2+)](i) was selectively increased by agonist stimulation of AMPA receptors. Unexpectedly, delayed neurotoxicity was attenuated at saturating doses of glutamate while little difference in cell viability was observed at lower doses or with the other excitotoxins at any concentration. Therefore, there was a dissociation between rises in AMPA receptor-mediated Ca(2+) influx and neurotoxicity despite marked decreases in GluR2 but not GluR1 immunoreactivity. It is proposed that a modification of AMPA receptor stochiometry that raises agonist-stimulated Ca(2+) influx during an excitotoxic insult may have eventual neuroprotective effects.     

Invited review: significance of spatial and temporal heterogeneity of calcium transients in smooth muscle.

The multiplicity of mechanisms involved in regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) in smooth muscle results in both intra- and intercellular heterogeneities in [Ca(2+)](i). Heterogeneity in [Ca(2+)](i) regulation is reflected by the presence of spontaneous, localized [Ca(2+)](i) transients (Ca(2+) sparks) representing Ca(2+) release through ryanodine receptor (RyR) channels. Ca(2+) sparks display variable spatial Ca(2+) distributions with every occurrence within and across cellular regions. Individual sparks are often grouped, and fusion of sparks produces large local elevations in [Ca(2+)](i) that occasionally trigger propagating [Ca(2+)](i) waves. Ca(2+) sparks may modulate membrane potential and thus smooth muscle contractility. Sparks may also be the target of other regulatory factors in smooth muscle. Agonists induce propagating [Ca(2+)](i) oscillations that originate from foci with high spark incidence and also represent Ca(2+) release through RyR channels. With increasing agonist concentration, the peak of regional [Ca(2+)](i) oscillations remains relatively constant, whereas both frequency and propagation velocity increase. In contrast, the global cellular response appears as a concentration-dependent increase in peak as well as mean cellular [Ca(2+)](i), representing a spatial and temporal integration of the oscillations. The significance of agonist-induced [Ca(2+)](i) oscillations lies in the establishment of a global [Ca(2+)](i) level for slower Ca(2+)-dependent physiological processes.     

Aluminum as a specific inhibitor of plant TPC1 Ca2+ channels

In plant cells, Al ion plays dual roles as an inducer and an inhibitor of Ca(2+) influx depending on the concentration. Here, the effects of Al on Ca(2+) signaling were assessed in tobacco BY-2 cells expressing aequorin and a putative plant Ca(2+) channel from Arabidopsis thaliana, AtTPC1 (two-pore channel 1). In wild-type cells (expressing only aequorin), Al treatment induced the generation of superoxide, and Ca(2+) influx was secondarily induced by superoxide. Higher Al concentrations inhibited the Al-stimulated and superoxide-mediated Ca(2+) influx, indicating that Ca(2+) channels responsive to reactive oxygen species (ROS) are blocked by high concentration of Al. H(2)O(2)-induced Ca(2+) influx was also inhibited by Al. Thus, inhibitory action of Al against ROS-induced Ca(2+) influx was confirmed. Similarly, known Ca(2+) channel blockers such as ions of La and Gd inhibited the H(2)O(2)-induced Ca(2+) influx. While La also inhibited the hypoosmotically induced Ca(2+) influx, Al showed no inhibitory effect against the hypoosmotic Ca(2+) influx. The effects of Al and La on Ca(2+) influx were also tested in the cell line overexpressing AtTPC1 and the cell line AtTPC1-dependently cosuppressing the endogenous TPC1 equivalents. Notably, responsiveness to H(2)O(2) was lost in the cosuppression cell line, thus TPC1 channels are required for ROS-responsive Ca(2+) influx. Data also suggested that hypoosmotic shock induces TPC1-independent Ca(2+) influx and Al shows no inhibitory action against the TPC1-independent event. In addition, AtTPC1 overexpression resulted in a marked increase in Al-sensitive Ca(2+) influx, indicating that TPC1 channels participate in osmotic Ca(2+) influx only when overexpressed. We concluded that members of TPC1 channel family are the only ROS-responsive Ca(2+) channels and are the possible targets of Al-dependent inhibition.     

Induction of a sodium ion influx by progesterone in human spermatozoa

In human spermatozoa, progesterone (P(4)) induces a depolarization of the plasma membrane, a rapid calcium (Ca(2+)) influx, and a chloride efflux. The sodium ion (Na(+)) was partly responsible for the P(4)-induced depolarizing effect but was not required for calcium influx. We used fluorescent probes for spectrofluorometry to investigate whether P(4) induced a Na(+) influx and whether voltage-operated channels were involved in Na(+) and/or Ca(2+) entries. We found that 10 microM P(4) significantly increased intracellular Na(+) concentration from 17.8 +/- 2.0 mM to 27.2 +/- 1. 6 mM (P < 0.001). Prior incubation of spermatozoa with 10 microM flunarizine, a Na(+) and Ca(2+) voltage-dependent channel blocker, inhibited the sodium influx induced by 10 microM P(4) by 84.6 +/- 15.4%. The Ca(2+) influx induced by 10 microM P(4) was also significantly inhibited in a Na(+)-containing medium by 10 microM flunarizine or 10 microM pimozide (P < 0.01). In contrast, flunarizine had no inhibitory effect on the Ca(2+) influx induced by 10 microM P(4) in spermatozoa incubated in Na(+)-depleted medium. The P(4)-promoted acrosome reaction (AR) was significantly higher when spermatozoa were incubated in Na(+)-containing medium as compared to Na(+)-depleted medium. These data demonstrate that P(4) stimulates a Na(+) influx that could be involved in the AR completion. They also suggest that voltage-dependent Na(+) and Ca(2+) channels are implicated in P(4)-mediated signaling pathway in human spermatozoa.     

Effect of lanthanum on ion absorption in corn roots

Short term (10 min) influx of (86)Rb-labeled potassium into corn (Zea mays L. WF9 x M14) root segments was inhibited by La (NO(3))(3) or LaCl(3). Half maximal inhibition of K(+) influx from 0.25 mm KCl was obtained with 0.025 mm La(3+). Kinetic analysis indicated the inhibition to be of a competitive nature. With absorption periods exceeding one hour, La(3+) no longer inhibited, but rather stimulated K(+) influx rates. La(3+) was not an inhibitor of (36)Cl or (32)P absorption. Separated cortex and stele absorbed labeled potassium (and phosphate) at comparable rates, and La(3+) inhibited potassium influx in both tissues. The effects of La(3+) on ion absorption were similar to those of Ca(2+), suggesting that the two polyvalent cations act at the same site. Based on this and the observation that La(3+) does not seem to penetrate the plasma membrane, it was concluded that La(3+) and Ca(2+) affect changes in ion transport without entering cells.     

白藜芦醇降低大鼠心室肌细胞内游离钙浓度

实验旨在研究白藜芦醇(resveratrol)对大鼠心室肌细胞内钙浓度(intracellular calcium concentratoin,[Ca2+]i)的影响.应用激光共聚焦显微镜技术记录心室肌细胞内的钙荧光强度.结果表明在正常台氏液和无钙台氏液中,白藜芦醇(15～60μmol/L)呈浓度依赖性地降低[Ca2+]i.蛋白酪氨酸磷酸酶抑制剂正钒酸钠(sodium orthovanadate,1.0 mmol/L)和L型Ca2+通道激动剂Bay K8644(10 μmol/L)可部分抑制正常台氏液中白藜芦醇的效应.但NO合酶阻断剂L-NAME(1.0 mmol/L)对白藜芦醇的作用无影响.白藜芦醇也能明显抑制无钙台氏液中由低浓度ryanodine(1.0 nmol/L)引起的[Ca2+]i增加.当细胞外液钙浓度由1 mmol/L增加到10 mmol/L而诱发心室肌细胞钙超载时,部分心室肌细胞产生可传播的钙波,白藜芦醇(60 μmol/L)可降低钙波的传播速度和持续时间,最终阻断钙波.结果提示,白藜芦醇能够降低心室肌细胞内游离钙浓度,此作用可能与其抑制电压依赖性Ca2+通道、酩氨酸激酶和肌浆网内钙释放有关.     

Steady-state currents through voltage-dependent, dihydropyridine-sensitive Ca2+ channels in GH3 pituitary cells.

Ca2+ influx via voltage-dependent Ca2+ channels is known to be elicited during action potentials but possibly also occurs at the resting potential. The steady-state current through voltage-dependent Ca2+ channels and its role for the electrical activity was, therefore, investigated in pituitary GH3 cells. Applying the recently developed 'nystatin-modification' of the patch-clamp technique, most GH3 cells (18 out of 23 cells) fired spontaneous action potentials from a baseline membrane potential of 43.7 +/- 4.6 mV (mean +/- s.d., n = 23). The frequency of action potentials was stimulated about twofold by Bay K 8644 (100 nM), a Ca(2+)-channel stimulator, and action potentials were completely suppressed by the Ca(2+)-channel blocker PN 200-110 (100 nM). Voltage clamping GH3 cells at fixed potentials for several minutes and with 1 mM Ba2+ as divalent charge carrier, we observed steady-state Ca(2+)-channel currents that were dihydropyridine-sensitive and displayed a U-shaped current-voltage relation. The results strongly suggest that the observed long lasting, dihydropyridine-sensitive Ca(2+)-channel currents provide a steady-state conductivity for Ca2+ at the resting potential and are essential for the generation of action potentials in GH3 pituitary cells.